In the past insulin has been synthezised (from synthetic A- and B-chains) or re-synthesized (from naturally derived A- and B-chains) by combining the two chains in an oxidation process whereby the 6 cysteine sulfhydryl groups of the reduced chains (4 in the A-chain, 2 in the B-chain) are converted into disulfide bonds. By this method disulfide bonds are formed largely at random, meaning that the yield of insulin with disulfide bridges correctly positioned between cysteine residues A-6 and A-11, A-7 and B-7, and A-20 and B-19, respectively, is very low.
Following the discovery of proinsulin as a biological precursor of insulin it was observed that the A- and B-polypeptide moieties of the linear-chain totally reduced proinsulin (those moieties corresponding to the A- and B-chains of insulin, respectively) could be oxidatively combined with much less randomization of the disulfide bonds to give a substantially higher yield of correctly folded proinsulin as compared with the combination of free A- and B-chains (D. F. Steiner et al.: Proc. Nat. Acad. Sci. 60 (1968), 622). Albeit high yields were obtained only at proinsulin concentrations too low to make the process feasible on a preparative scale, the function of the C- (i.e. connecting peptide) moiety of the B-C-A polypeptide sequence of proinsulin, namely that of bringing the 6 cysteine residues into spatial positions favorable for correct oxidation into proinsulin, was clearly demonstrated.
The proinsulin formed may function as an in vitro precursor of insulin in that the connecting peptide is removable by enzymatic means (W. Kemmler et al.: J. Biol. Chem. 246 (1971), 6786).
Subsequently it has been shown that proinsulin-like compounds having shorter linking moieties than the C-peptide and flanked at both ends by specific enzymatic or chemical cleavage sites (the so-called miniproinsulins (A. Wollmer et al., Hoppe-Seyler's Z. Physiol. Chem. 355 (1974), 1471-1476 and Dietrich Brandenburg et al., Hoppe-Seyler's Z. Physiol. Chem. 354 (1973), 1521-1524)) may also serve as insulin precursors.
Endeavours to provide biosynthetic insulins, particularly that identical to the human species, have followed the same strategic pathways as those to synthetic insulin. The insulin A- and B-chains have been expressed in separate host organisms, isolated therefrom and then combined as described supra (R. E. Chance et al.: Diabetes Care 4 (1982), 147). Microorganisms have been transformed with cloning vectors encoding preproinsulin or proinsulin which may be secreted as such (W. Gilbert et al.: European Patent Publ. No. 6694) or accumulated intracellularly as hybrid gene products (D. V. Goeddel et al.: European Patent Publ. No. 55945). The miniproinsulin pathway has also been attempted (D. V. Goeddel, supra).
Procuring the A- and B-chains in separate fermentation processes followed by combination of the chains is inherently impractical. The dual fermentation inconvenience may be overcome by choosing the proinsulin or miniproinsulin strategy. However, the use of a proinsulin as the biosynthetic insulin precursor may entail certain disadvantages. The proinsulin, whether excreted into the fermentation liquid as such or accumulated intracellularly in the host organism, possibly as a hybrid gene product, is likely to contain substantially randomized disulfide bonds. The refolding of such "scrambled" products into correctly folded proinsulin may be conducted either directly (H.--G. Gattner et al.: Danish Patent Application No. 4523/83) or via the single chain hexa-S-sulfonate (F. B. Hill: European Patent Publ. No. 37255). The refolding process usually entails some degree of polymerization and hence the inconvenience of using laborious purification steps during recovery.
In addition, insulin precursors of the proinsulin type are prone to undergo enzymatic degradation, either within the host cells or following its excretion into the fermentation broth. In yeast it has been shown that human proinsulin is particularly sensitive to enzymatic cleavages at the two dibasic sequences (Arg31-Arg32 and Lys64-Arg65). Apparently these cleavages occur before the establishment of the S--S bridges, resulting in the formation of C-peptide, A-chain and B-chain.